Increased expression of protein disulphide isomerase (PDI), particularly PDIA3, is associated with breast cancer cell aggressiveness. However, it has not been explored whether PDIA3 modulates cancer cell phenotypes by altering cancer cell metabolism. Here, we investigated the effects of C-3399, a novel PDIA3 inhibitor, on the adhesion of breast cancer cells to the extracellular matrix (ECM) and pulmonary microvascular endothelial cells (hLMVEC). Additionally, we explored whether the anti-adhesive effect of PDIA3 inhibition by C-3399 could be mediated by changes in cellular bioenergetics. We found that PDIA3 inhibition modifies adhesive interactions of two human breast cancer lines, representing the luminal (MCF-7) and basal (MDA-MB-231) subtypes, to ECM and hLMVEC. We confirmed that the anti-adhesive effect of C-3399 was due to the inhibition of PDIA3, as the effect was lost in cancer cells with silenced PDIA3. MCF-7 and MDA-MB-231 cells displayed distinct metabolic profiles, with higher levels of tricarboxylic acid (TCA) cycle metabolites in MCF-7. Interestingly, the anti-adhesive effect of PDIA3 inhibition was associated with the downregulation of TCA metabolites (malate, fumarate, alpha-ketoglutarate, isocitrate) and increased lactate production, particularly in MCF-7 cells. Treatment with mitochondrial respiration inhibitors phenocopied the anti-adhesive effect in MCF-7 but had weaker effects in MDA-MB-231 cells. Quantification of C-3399 and its major metabolite (C-3399-B) revealed the extracellular metabolism of the active compound. In conclusion, the inhibition of extracellular PDIA3 represents a novel approach to inhibit the mitochondrial bioenergetic metabolism of cancer cells and to limit adhesion of cancer cells to the pulmonary endothelium.